TORONTO — The U.S. Drug Enforcement Administration is investigating a Mexican businessman who supplied the plane that flew Canadian mediator Cynthia Vanier and a group of former Special Forces soldiers on a controversial Libyan mission last year.

The DEA has subpoenaed witnesses to testify next week as part of a probe of Christian Esquino, who rented Ms. Vanier the jet that transported her and her entourage to North Africa last July during the NATO-backed revolt that ousted dictator Col. Muammar Gaddafi.

The U.S. investigation is not directly related to Ms. Vanier’s expedition, which was financed by Montreal-based SNC-Lavalin but which Mexican authorities allege was part of a plot to smuggle Col. Gaddafi’s son Saadi to Mexico using falsified passports. Ms. Vanier and three alleged co-conspirators are currently imprisoned and awaiting trial in Mexico on charges related to the alleged smuggling scheme.

But the renewed interest in Mr. Esquino may have been triggered by his link to the high-profile Libya case. It also raises questions about Mr. Esquino’s role as a key Mexican government witness in the Gaddafi smuggling case.

Hints that the DEA was pursuing Mr. Esquino surfaced in February, when the drug agency seized one of his jets after it landed at a Tuscon airport from Mexico. Last week the agency began ordering witnesses to appear before a DEA special agent to give testimony under oath.

Among those subpoenaed was Mr. Esquino’s sister-in-law, Norma Gonzalez, manager of Starwood Management. Starwood was the company Ms. Vanier’s firm, Vanier Consulting, contracted to supply the jet and pilots that flew her overseas for her Libyan mission.

The subpoena ordered Ms. Gonzalez to appear at the DEA office in San Diego on Monday and to bring all company records “showing the business relationship” between Starwood, Mr. Esquino, Veritas Worldwide Security Inc. (the California airplane broker that arranged the jet) and Gabriela Cueto, Ms. Vanier’s co-accused.

The DEA subpoena also asked for Starwood’s emails and payment records related to several other companies and individuals, including the Grammy-winning Guatemalan pop singer Edgar Ricardo Arjona Morales and Jorge Hank-Rhon, the colorful former mayor of Tijuana and one of Mexico’s richest men.

Mr. Hank-Rhon runs a Mexican gambling empire called Grupo Caliente and has long faced allegations of ties to organized crime and the Tijuana drug cartel. But he has repeatedly refuted the accusations. Last June, Mexican authorities raided his compound, seizing 88 weapons and more than 90,000 rounds of ammunition. He was held briefly on charges related to weapons and ordering a murder but was soon released for lack of evidence.

Mr. Esquino, also known as Ed Nunez, denies any involvement in drug smuggling or money laundering. He went through a similar probe a decade ago, when he was investigated for alleged links to the Tijuana drug cartel. He was never charged and was deported to Mexico after serving a two-year fraud sentence.

In January, he testified to Mexican authorities that Ms. Vanier had hired his planes to fly Saadi Gaddafi to Mexico. Ms. Vanier has denied any involvement and says her Libyan trip was a fact-finding mission for SNC-Lavalin, which has several construction projects in the country.

In an email sent in April to Gregory Gillispie, the owner of Veritas and Ms. Cueto’s business associate, Mr. Esquino took issue with comments Mr. Gillispie had made to reporters about the Libya affair.

“A little piece of information that might persuade you to keep your mouth shut and prove to everyone that you’re a liar: I secretly recorded you and Gabby [Ms. Cueto] for the Mexican government … talking about the fact that we were going to fly Saadi Gaddafi and other members of their clan to Mexico.”

But Mexican prosecutors have not publicly acknowledged the existence of any such tape and Mr. Gillispie responded to Mr. Esquino with an email that said he hoped the recordings were clear. “If they were then it would prove conclusively that I never asked you to fly Saadi Gaddafi or his clan to Mexico.”